Human monoclonal antibody against the VP1 protein of JC virus

Abstract

A human neutralizing monoclonal antibody is directed against the VP1 protein of JC virus. The JC virus is responsible for progressive multifocal leukoencephalopathy (PML). The antibody is used in a therapeutic or prophylactic treatment of a JCV infection or of a disease associated with a JCV infection, such as progressive multifocal leukoencephalopathy (PML). The antibody is also used in the diagnosis of JCV infections or of diseases associated with JCV infections.

Claims

1. A recombinant human monoclonal antibody specifically binding VP1 protein of JC virus, wherein the monoclonal antibody is capable of neutralizing the JC virus, the human monoclonal antibody comprising at least a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the sequence SEQ ID NO:1 or is encoded by the sequence SEQ ID NO:3 and the light chain variable region has the sequence SEQ ID NO:2 or is encoded by the sequence SEQ ID NO: 4.

2. The monoclonal antibody according to claim 1, which is a full-length immunoglobulin or an antigen-binding fragment thereof selected from the group consisting of Fab, Fab, F(ab).sub.2, Fv, and single chain antibodies (scFv).

3. The monoclonal antibody according to claim 1, for therapeutic or prophylactic treatment of a JCV infection or progressive multifocal leukoencephalopathy (PML) associated with a JCV infection.

4. A pharmaceutical composition comprising a monoclonal antibody according to claim 1.

5. The pharmaceutical composition according to claim 4 for the therapeutic or prophylactic treatment of a JCV infection or progressive multifocal leukoencephalopathy (PML) associated with a JCV infection.

6. An in vitro method of diagnosis of a JCV infection or progressive multifocal leukoencephalopathy (PML) associated with a JCV infection, the method comprising the steps of: contacting a biological sample from a patient suspected of being infected with JCV with a monoclonal antibody according to claim 1, under suitable conditions for the monoclonal antibody to bind the JCV VP1 protein, if present in the sample, and qualitatively or quantitatively detecting the binding of the monoclonal antibody to the JCV VP1 protein, said binding being indicative of JCV infection or progressive multifocal leukoencephalopathy (PML) associated with a JCV infection.

7. The method according to claim 6, wherein detecting is performed with an immunological ELISA assay or an immunological fluorescent assay or an immunohistochemical assay.

8. An immunodiagnostic kit for the diagnosis of a JCV infection or progressive multifocal leukoencephalopathy (PML) associated with a JCV infection, the kit comprising a recombinant human monoclonal antibody according to claim 1 and instructions for carrying out an in vitro immunodiagnostic method.

9. An immunodiagnostic kit, the kit comprising a recombinant human monoclonal antibody directed against the VP1 protein of JC virus, wherein the monoclonal antibody is a human antibody and is capable of neutralizing the JC virus, and instructions for carrying out an in vitro immunodiagnostic method, wherein the in vitro immunodiagnostic method is a method according to claim 6.

10. A method of neutralizing the JCV virus in a human patient suffering from a JCV infection, the method comprising administering to a human patient an effective amount of a monoclonal antibody according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph illustrating the percentages of the anti-JCV neutralizing activity of sera that had previously demonstrated by ELISA a reactivity >1 O.D. 450 towards VP1.

(2) FIG. 2 shows the results of a Western Blot assay under denaturing conditions performed with the JCV VP1 protein stained with the murine antibody Abcam and the human monoclonal antibody GRE.

(3) FIG. 3 shows the results of a Dot Blot assay performed both with denatured VP1 and wild-type VP1, stained with the murine antibody Abcam and the human monoclonal antibody GRE.

(4) FIG. 4 is a bar graph showing the binding percentages of the murine antibody Abcam and the human monoclonal antibody GRE to JCV VP1 protein subjected to alanine scanning site-directed mutagensis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Example 1

(5) A combinatorial phage display library of human antibody fragments (monovalent Fabs), of IgG1/k isotype and with an estimated size of 210.sup.7 elements, was constructed in the pPD phagemid vector through methods similar to those previously described (Plaisant, P., et al., Human monoclonal recombinant Fabs specific for HCV antigens obtained by repertoire cloning in phage display combinatorial vectors. Res Virol, 1997. 148(2): p. 165-9). The library was generated from the bone marrow of a 68 year old man whose serum had been tested positive for the presence of anti-VP1/JCV antibodies by ELISA. The ELISA was performed by coating a 96-well plate with 100 ng/well of recombinant JCV VP1 protein (Mad1, Abcam) in phosphate buffered saline (PBS). Several serum dilutions were added in duplicate to a VP1-coated plate. The plate was incubated at 37 C. for 1 hour and then washed with 0.1% PBS/TWEEN 20. The bound antibodies were detected with an anti-human IgG1 antibody conjugated with peroxidase (HRP) (Sigma-Aldrich).

(6) The biopanning of the phage-expression combinatorial antibody library for selecting the anti-VP1 antibody was carried out as previously described (Williamson, R. A., et al., Human monoclonal antibodies against a plethora of viral pathogens from single combinatorial libraries. Proc Natl Acad Sci USA, 1993. 90(9): p. 4141-5). Briefly, a phage preparation (0.1 ml/well) at a concentration of 10.sup.12 phages per milliliter was used in each of the biopanning cycles for selecting the antibody on high-binding ELISA plates (Costar) coated with VP1 from Mad1 strain. A total of 5 biopanning cycles were carried out and the phages obtained from the last 3 cycles were converted into a phagemid system expressing soluble Fabs.

(7) Escherichia coli cells from the XL-1-blue strain (Agilent Technologies), transformed with a Fab expression vector, were used for producing the selected Fab molecules for further characterization. Briefly, Fab preparations were obtained by freeze-thaw procedures from a culture. Five ml of SB (super broth) containing ampicillin (50 g/ml; Sigma-Aldrich) were inoculated with the transformed bacteria and grown for 7 hours at 37 C. in a rotating stirrer. Isopropyl--D-thiogalactopyranoside (IPTG, 1 mmol/l; Sigma-Aldrich) was added to the growing bacteria, which were further incubated overnight at 30 C. Then, the cells were centrifuged, resuspended in 1 ml of PBS/1% bovine serum albumin (BSA), and subjected to a freeze-thaw procedure (3 rounds). The cell debris was reduced to a pellet by centrifugation at 13000 g at room temperature in a microfuge, and the supernatant was used in an ELISA assay with no further processing. The ELISA was performed as previously described.

(8) Clones that yielded OD.sub.450>0.8 by ELISA against JCV VP1 were considered positive and further characterized.

(9) 105 clones were analyzed, 11 clones tested positive (10.5%). No reactivity was demonstrated against BSA.

(10) In order to confirm the specificity of the positive clones by ELISA, these were tested by an immunofluorescence assay on COS-7 cells (Transformed African Green Monkey Kidney Fibroblast Cells) infected with the Mad4 JCV strain (the recombinant VP1 protein from Mad1 strain was used in the ELISA). A goat anti-human Fab antiserum conjugated with fluorescein isothiocyanate (Sigma-Aldrich) was used as the secondary antibody. All the ELISA-tested positive clones were confirmed capable of binding to infected cells, whereas no reactivity was detected with uninfected cells. The ELISA-tested negative clones were not able to bind to infected cells.

(11) The nucleic acid from the ELISA-tested positive clones was obtained with the Spin Miniprep kit (Qiagen) and was sequenced on a 373A sequencer (PerkinElmer). For sequencing the heavy chain, the primer SEQGz (5-GTCGTTGACCAGGCAGCCCAG-3) (SEQ ID NO:5), which binds to the (+) strand, was used. For the light chain, the primer SEQKb (5-ATAGAAGTTGTTCAGCAGGCA-3) (SEQ ID NO:6), which binds to the (+) strand, was used. The sequence analysis was carried out with the BLAST and IMGT tools and showed that all the clones derive from the same subfamily of VH genes for the heavy chain, and Vk genes for the light chain, and share the same sequence. Furthermore, the DNA sequence of the antibody is new. It is also important to point out that in literature no human antibody has been so far described that is directed against JCV VP1 protein. Therefore, this is the first human anti-JCV VP1 monoclonal antibody to be described. This antibody has been identified by the name GRE1.

(12) In order to test the neutralizing activity of the human anti-VP1 GRE1 antibody, 510.sup.4/well COS-7 cells were seeded in complete DMEM medium in a 24-well plate (Corning). COS-7 are cells permissive to infection by JCV.

(13) The following day, approximately 100 focus forming units of JCV (Mad4 strain) were added to 100 l of progressive dilutions of GRE1 (two-fold dilutions, from about 20 g/ml to 0.3 g/ml). The mixture was incubated for 1 hour at 37 C. and then added to COS-7 cells. Afterwards, they were incubated for 2 hours at 37 C. After one wash with PBS, 500 l of fresh medium were added to each well and the cells were incubated for 6 days at 37 C.

(14) Assessment of the Neutralizing Activity

(15) The neutralizing activity of the antibodies was assessed by immunofluorescence. Briefly, the immunofluorescence was carried out at 6 days post-infection. The slides were prepared by fixing the cells for 15 min with a Methanol/Acetone mixture (1:1 ratio) at room temperature and by using the commercial Abcam murine anti-VP1 antibody (1 g/ml) as the primary antibody, and the FITC-conjugated murine anti-Fab (Sigma-Aldrich) as the secondary antibody, following the guidelines of the manufacturer.

(16) The assessment is done by comparing the numbers of positive cells in the wells in which the virus was added to GRE1, with cells infected in the absence of the antibody (100% infection).

(17) The data observed under the fluorescence microscope were also confirmed by a robotized fluorescence reading system (IN Cell Analizer Sistem 1000, GE Healthcare) capable of automatically distinguishing the positive cells from the background, which indicated that the antibody as a Fab fragment was capable of inhibiting by more than 50% JCV infection at a concentration of 1 ng/l.

(18) Analogous experiments have also experimentally demonstrated that not all the sera from patients reactive against JCV VP1 are able to neutralize the virus.

(19) To that end, about 100 sera were tested by ELISA at a 1:400 dilution (dilution in PBS/1% BSA) to verify the presence of anti-JCV antibodies. In short, the ELISA plate (Costar) was covered with 25 l/well of a solution containing 100 ng of recombinant VP1 (Abcam) and incubated at 4 C. overnight. The following day, the plate was washed with water and blocked with PBS-1% BSA (w/v) for 1 hour at 37 C. Afterwards, 40 l of a single dilution (1:400 in PBS/1% BSA) of the serum to be tested were added and the plate was then incubated for 1 hour at 37 C. After having done 5 washes with PBS-0.1% Tween 20 (Sigma-Aldrich), by an automatic washer for ELISA microplates (ETI-System Kasher, DiaSorin), 40 l/well of the commercial horseradish peroxidase-conjugated anti-human IgG antibody were added (Sigma-Aldrich), following the indications of the manufacturer. The plate was then incubated for 45 minutes at 37 C. After having done several washes, as previously described, 40 l of substrate (1:1 solution of H.sub.2O.sub.2 and 3,3,5,5-tetramethylbenzidine, TMB substrate kit, Thermo Scientific) were added to each well, for the enzymatic reaction to occur. After about 15 minutes, the enzymatic activity was blocked by adding 40 l/well of 1N H.sub.2SO.sub.4 (Carlo Erba) and the colorimetric reaction was measured with a spectrophotometer (Model 680 Microplate Reader, Bio-Rad) at a wavelength of 450 nm.

(20) BSA antigen or another suitable antigen was introduced in each experiment as a negative control, whose O.D..sub.450 was used for detecting a possible non-specific reactivity.

(21) A few sera, which showed reactivity against JCV VP1>1 O.D. 450, were analyzed with a neutralization assay. Briefly, the day before infection, 510.sup.4/well COS-7 cells (permissive to infection by JCV) were seeded in complete DMEM medium in a 24-well plate (Corning). The following day, 200 l of the JCV (Mad1)-containing medium were added to 200 l of the 1:200 dilution of the serum to be tested (the final dilution of the serum is then 1:400, the same dilution used for the ELISA assay). The mixture was incubated for 1 hour at 37 C., and then added to the COST cells. Afterwards, they were incubated for 2 hours at 37 C. After one PBS wash, 500 l of fresh medium were added to each well and the cells were incubated for 6 days at 37 C.

(22) The neutralizing activity of the tested samples was assessed by indirect immunofluorescence. The slides were prepared by fixing the cells for 15 min with a Methanol/Acetone mixture (1:1 ratio) at room temperature and by using the commercial Abcam murine anti-VP1 antibody (1 g/ml) as the primary antibody, and the FITC-conjugated murine anti-IgG (Sigma-Aldrich) as the secondary antibody, following the guidelines provided by the manufacturer.

(23) The assessment was done by comparing the numbers of positive cells in the wells in which the virus was added to the tested sample, with cells infected in the absence of the sample (100% infection).

(24) This analysis demonstrated that, in spite of the reactivity towards VP1 by ELISA, a non negligible number of sera showed no neutralizing anti-JCV activity. The obtained results are illustrated in the graph in FIG. 1. In the graph of FIG. 1, the percentages of the neutralizing activity of some of the tested sera are reported. All the reported sera demonstrated by ELISA a reactivity >1 O.D.450 towards VP1.

(25) Immunofluorescence and Immunohistochemistry on Biopsy Samples

(26) By using the anti-JCV VP1 GRE1 antibody of the present invention in immunofluorescence and immunohistochemistry assays, the presence of JCV can be determined in biopsy samples. This antibody actually showed high sensitivity and specificity for JCV VP1 protein, while on the contrary showed no reactivity towards BKV VP1.

(27) The biopsy samples may be fresh or fixed or paraffinized samples. In the case of fixed and paraffinized samples, if necessary, the sample is deparaffinized and the antigenicity is restored according to standard procedures. The sample is then incubated at 37 C. for 30 minutes with GRE1 (10 g/ml in PBS). After the incubation period, the sample is washed 5 times in PBS and then incubated for 30 min at 37 C. with the FITC- or horseradish peroxidase-conjugated anti-human Fab (Sigma-Aldrich), following the guidelines of the manufacturer. In case of use of the antibody conjugated with horseradish peroxidase, it is necessary to add the substrate (diaminobenzidine, DAB substrate kit, Thermo Scientific), which in the presence of peroxidase produces a brown precipitate that allows to visualize the possible antibody binding.

(28) The evaluation of the assay is carried out by observing the sample under a fluorescence microscope or with an automated fluorescence detection system if the FITC-conjugated antibody is used, or by light microscopy or with an automated imaging system if the horseradish peroxidase-conjugated antibody is used.

(29) Capture ELISA for Detecting JCV in Biological Samples

(30) For carrying out an ELISA assay with the human anti-JCV VP1 GRE1 antibody, an ELISA plate (Costar) is covered with 25 l/well of a solution containing 40 ng of the sheep anti-human Fd antibody in PBS (1.6 g/ml final concentration) and incubated at 4 C. overnight. The following day, the plate is washed with water and blocked with PBS-1% BSA (w/v) for 1 hour at 37 C. Afterwards, 40 l of GRE1 (final concentration approximately 4 ng/l in PBS/1% BSA) are added per well and the plate is then incubated for 1 hour at 37 C. After having done 5 washes with PBS-0.1% Tween 20 (Sigma-Aldrich), by an automatic washer for ELISA microplates (ETI-System Kasher, DiaSorin), 40 l of several dilutions of the biological sample to be tested (serial 10-fold dilutions, starting from the undiluted up to 1:1000 dilutions) are added to each well. The plate is then incubated for 1 hour at 37 C. After having done several washes, as previously described, 40 l of a 1:1000 dilution of a commercial murine anti-VP1 antibody (Abcam diluted in PBS/BSA) are added to each well. The plate is left resting for 1 hour at 37 C. After further washes, 40 l/well of a polyclonal preparation of goat antibodies are added, which bind the Fc portion of murine IgG and are conjugated with horseradish peroxidase (Anti-Mouse IgG (Fc specific)-Peroxidase antibody produced in goat, Sigma-Aldrich). The plate is incubated for 45 minutes at 37 C. After 5 washes with PBS-Tween20 carried out as previously described, 40 l of substrate (1:1 solution of H.sub.2O.sub.2 and 3,3,5,5-tetramethylbenzidine, TMB substrate kit, Thermo Scientific) are added to each well, for the enzymatic reaction to occur. After about 15 minutes, the enzymatic activity is blocked by adding 40 l/well of 1N H.sub.2SO.sub.4 (Carlo Erba) and the colorimetric reaction is measured with a spectrophotometer (Model 680 Microplate Reader, Bio-Rad) at a wavelength of 450 nm.

(31) BSA antigen or another suitable antigen is introduced in each experiment as a negative control, whose OD.sub.450 is used for detecting a possible non-specific reactivity.

Example 2

Definition of the (Linear or Conformational) Nature of the Epitope Recognized by GRE1 Monoclonal Antibody

(32) In order to define the (linear or conformational) nature of the epitope recognized by GRE1 monoclonal antibody, Western Blot and Dot Blot assays were carried out both with the denatured protein and the wild type protein.

(33) FIG. 2 shows the results of the Western Blot assay under denaturing conditions. The protein was denatured with -mercaptoethanol (-mer) or with sodium dodecyl sulfate (SDS). The commercial murine antibody is designated as Abcam, whereas GRE1 anti-JCV VP1 monoclonal antibody is designated as GRE.

(34) FIG. 3 shows the results of the Dot blot assay performed both with denatured VP1 and VP1 in the wild type conformation. The commercial murine antibody is designated as Abcam, whereas GRE1 anti-JCV VP1 monoclonal antibody is designated as IgG GRE.

(35) The results show that GRE1 is not able to bind to the denatured form of the protein, but that it is only capable of recognizing the non-denatured protein. In corroboration of this fact, a commercial murine antibody (Abcam, ab34756) directed against a VP1 linear epitope, instead, was able to recognize both of the protein forms. Thus, these results lead to conclude that the epitope recognized by GRE1 monoclonal antibody is a conformational epitope.

(36) Definition of the Specificity of GRE1 Monoclonal Antibody

(37) The high nucleotide sequence homology (approximately 70%) between the BK virus genome (BKV) and the JCV genome, which are the main polyomaviruses capable of infecting man, has been extensively described in the literature.

(38) In order to determine if GRE1 is able to recognize JCV exclusively, its reactivity was tested by an ELISA assay against a recombinant JCV VP1 protein (Abcam, ab74569) and against the recombinant BKV VP1 protein (Abcam, ab74567). GRE1 proved to be capable of binding only to the JCV VP1 protein, pointing out its absolute specificity.

(39) Characterization of the Epitope Recognized by GRE1 Monoclonal Antibody by Alanine Scanning Site-Directed Mutagenesis

(40) The amino acid sequences of the JCV VP1 and BKV VP1 proteins were aligned by the ClustalX program, so as to identify the portions bearing the residues that show a profound difference (such as charge and polarity) between the two proteins and that may be responsible for the binding difference that the antibody of the invention has towards the two proteins. 33 amino acid residues completely different (as charge and polarity) between the JCV and BKV VP1s were identified through this analysis.

(41) In order to define the critical VP1 residues involved in the binding with GRE1 monoclonal antibody, the previously identified residues were individually mutated into alanine (or into glycine, in the case that the original residue was an alanine). Briefly, the site-directed mutagenesis was performed on the pcDNA 3.1 expression vector/V5-His TOPO TA Expression Kit (Life Technologies) where the nucleotide sequence encoding the VP1 protein from the Mad1 strain JCV had been previously cloned. The primers used for the mutagenesis were designed to be approximately 30 nucleotides in length and show a 15-20 nucleotide overlapping region at the 5 terminus, in such a way as to obtain an effective mutagenesis product. After the amplification reaction, the PCR product was digested with the enzyme DpnI for 4 hours at 37 C., so as to eliminate the methylated DNA used as the template. After digestion, 1 L of amplification was used to transform electro-competent cells. A few transformed colonies were isolated and checked by sequencing, in order to analyze if the desired mutation was inserted. Mutated VP1 was then cloned in another expression vector (pCAGEN, Addgene #11160).

(42) HEK 293T (human epithelial kidney) cells were transfected with the pCAG-VP1mut vectors, and the binding of the anti-VP1 antibody to these mutated VP was assessed by FACS (Fluorescence activated cell sorter). In short, HEK293T cells were transfected with 4 g of vector where mutated VP1 had been cloned. After centrifugation and fixation with 4% paraformaldehyde, the transfected cells were incubated for 30 minutes at room temperature with GRE1 or the Abcam commercial antibody directed against a C terminus linear sequence, diluted in a permeabilizing solution at a concentration of 1 g/ml. The cells were then washed and incubated for 30 minutes at room temperature with anti-human or anti-mouse FITC-conjugated monoclonal antibodies, following the indications of the manufacturer (Sigma-Aldrich) and thereafter analyzed by FACS. The reactivity observed with the non-mutated VP1 protein was considered as 100% binding, instead non-transfected cells were used as the negative control.

(43) The software GraphPad Prism was used for the analysis of the data obtained by FACS and for the graphic editing.

(44) By FACS analysis, it was observed that the residues which, if mutated, disrupt the binding are: I62A, S65A, A127G, D130A, N131A, A133G, A175G (amino acid numbering based on the residues of the Mad1 strain VP1, with the numbering starting from Met at position 1) (FIG. 4). Instead, mutated residues capable of changing the conformation of the protein or drastically decreasing the expression thereof in vitro were not considered in this analysis.

(45) The crystallographic model already described previously in the literature and filed in the free-access RCSB-PDB databank (www.rcsb.org), with the accession code 3NXG (www.rcsb.org/pdb/explore/explore.do?structureId=3NXG) was used for the structural characterization of the epitope recognized by GRE1. The use of this model allowed to notice that residues that are far away on the single monomer, on the contrary are very close on two adjacent monomers in the pentameric form and absolutely contiguous to the portion of the protein involved in the binding with sialic acid. These structural data explain the considerable neutralizing activity of GRE1.

(46) The obtained experimental data show that GRE1 recognizes a conformational, not a linear, epitope comprising at least the following residues: I62, S65, A127, D130, N131, A133, A175 (amino acid numbering based on the residues of the Mad1 strain VP1, with the numbering starting from Met at position 1).

(47) Particularly, it was found that the residues, that if mutated disrupt the binding of GRE1, are very close to the region that is important for the binding between VP1 protein and its receptor, and this explains the neutralizing activity of GRE1.

(48) ELISA Assay for the In Vitro Determination of the Presence of Neutralizing Antibodies in Biological Samples

(49) In order to determine the presence of neutralizing antibodies in biological samples (particularly, but not exclusively, in cerebrospinal fluid, serum, or plasma), the epitope recognized by GRE1 (neutralizing antibody) was used in an ELISA assay. The setting-up of a fast and effective detection system was done with two strategies: one of them in which a competitive ELISA assay is carried out with the samples and GRE1, the other one in which the least portion of VP1 (comprising the residues between the positions 50 and 140 of the original protein) that can still be recognized by the neutralizing antibody is used as the antigen. A variant of the latter assay was also set up, wherein the biological samples to be tested were made to compete in the liquid phase against a VP1 that is only mutated in the residues recognized by GRE1, so as to more effectively subtract from the reaction the antibodies capable of recognizing the VP1 portions that are different from those recognized by GRE1.

(50) ELISA Assay for the In Vitro Determination of the Presence of Neutralizing Antibodies by Competition for the VP1 Protein with GRE1 Monoclonal Antibody

(51) In order to distinguish GRE1 from the other human antibodies in the biological sample to be tested, GRE1 was labeled by methods of gene expression fusion with the amino acid sequence DYKDDDDK (SEQ ID NO: 12), so as to be exclusively recognized by the commercial monoclonal antibody FLAG M2 conjugated with horseradish peroxidase (Sigma-Aldrich).

(52) The ELISA plate (Costar) was covered with 25 L/well of a solution containing 300 ng of the recombinant VP1 (Abcam, ab74569) and incubated at 4 C. overnight. The following day, the plate was washed with water and blocked with PBS-1% BSA (w/v) for 1 hour at 37 C. Afterwards, 40 L of several dilutions (serial 10-fold dilutions, starting from the undiluted up to 1:1000 dilutions) of the biological sample to be tested were added and the plate was then incubated for 1 hour at 37 C. Thereafter, 40 l of the GRE1 anti-JCV VP1 antibody (final concentration approximately 1 ng/L in PBS/1% BSA) were added to each well with the various sample dilutions and the plate was incubated for 30 minutes at 37 C. After having done 5 washes with PBS-0.1% Tween 20 (Sigma-Aldrich), by an automatic washer for ELISA microplates (ETI-System Kasher, DiaSorin), 40 L/well of the commercial horseradish peroxidase-conjugated FLAG M2 antibody were added (Sigma-Aldrich). The plate was then incubated for 45 minutes at 37 C. After having done several washes, as previously indicated, 40 L of substrate (1:1 solution of H.sub.2O.sub.2 and 3,3,5,5-tetramethylbenzidine, TMB substrate kit, Thermo Scientific) were added to each well, for the enzymatic reaction to occur. After about 15 minutes, the enzymatic activity was blocked by adding 40 pt/well of 1N H.sub.2SO.sub.4 (Carlo Erba) and the colorimetric reaction was measured with a spectrophotometer (Model 680 Microplate Reader, Bio-Rad) at a wavelength of 450 nm.

(53) BSA antigen or another suitable antigen was introduced in each experiment as a negative control, whose O.D..sub.450 was used for detecting a possible non-specific reactivity.

(54) The difference in absorbance detected in the wells where the monoclonal antibody was added with the different dilutions of the biological sample to be tested and in the wells where the monoclonal antibody was added alone was observed in order to estimate the inhibition of GRE1 binding.

(55) Particularly, if the absorbance in the wells where the monoclonal antibody was added with the different dilutions of the biological sample is lower than the absorbance observed with the monoclonal antibody alone, there has been a competition for epitope binding on VP1 between GRE1 and the antibodies in the sample, thereby indicating the presence of JCV-GRE1-like antibodies in the sample, and thus the presence of neutralizing and protective antibodies.

(56) ELISA Assay for the In Vitro Determination of the Presence of Antibodies in Biological Samples Capable of Recognizing the Same Epitope Bound by GRE1 Monoclonal Antibody

(57) In order to determine the minimal portion of VP1 that contains the epitope recognized by GRE1 monoclonal antibody, and yet retains the conformational features, the nucleotide sequence encoding for VP1 from Mad1 strain JCV was digested with DNases so as to obtain randomly cut VP1 sequences. The various VP1 portions were cloned in a phagemid in order to select, through bio-panning against GRE1, the VP1 portion still recognized by the monoclonal antibody. Once the portion still recognized by GRE1 was determined, the fragment was analyzed by specific software to determine the conformation in silico and possibly compare it with the conformation of the full-length VP1 protein.

(58) Taking into consideration the results of this prediction, the selected nucleotide sequence encoding the minimal portion recognized by GRE1, and comprising the residues between positions 50 and 140 of the original protein, was cloned in the bacterial expression vector pET15b, in frame with 6His-tag and a thrombin cleavage site (pETminiVP1). The restriction sites XhoI and BamHI (present in the vector cloning region, but not within VP1) were used for the cloning. To that end, the VP1 region of interest was amplified by PCR, by inserting respectively the XhoI and BatnHI restriction sites at the 5 and the 3. The selection of the colonies that contained pETminiVP1 was done on the basis of ampicillin resistance and by sequence analysis.

(59) For the purification of the fragment, one pETminiVP1-containing colony was inoculated in 10 ml of LB with 50 g/mL ampicillin and grown at 37 C. overnight. The following day, 5 ml of culture were sub-inoculated in 500 ml of LB medium with 50 g/mL ampicillin. The culture was analyzed at regular intervals with a spectrophotometer to check the bacterial growth. When the culture reached an OD600 of 0.6-1, 0.4 mM isopropyl--D-1-thiogalactopyranoside (IPTG) was added to the medium and left stirring at room temperature overnight.

(60) The following day, the bacterial culture was centrifuged for 15 minutes at 3900 rcf, and the bacterial pellet was resuspended in 20 ml of buffer A (50 mM Tris pH 7.5, 5% glycerol, 250 mM NaCl, 30 mM imidazole). The cells were then lysed by using a sonicator. The suspension was centrifuged for 45 minutes at 12,000 rcf and filtered with 0.4 m filters to eliminate the bacterial debris.

(61) The fragment was then purified by affinity chromatography on a nickel column. Before applying the sample to the column, the resin was washed with 3 volumes of buffer A, 3 volumes of buffer B (20 mM Tris pH 7.5, 5% glycerol, 250 mM NaCl, 500 mM Imidazole) and re-equilibrated with 8 volumes of buffer A. At this time, the sample was run on the column. 50 ml of buffer A were used to wash the column from the unbound sample, and the sample was eluted with a 0%-100% gradient of buffer B. After elution, the column was re-equilibrated with buffer A. Once concentrated, the fragment was digested with thrombin so as to eliminate the histidine tail that could affect the biological sample to be tested. In order to eliminate traces of the elution buffer that could affect a possible use of the purified fragment, the elution was dialyzed against PBS. The quality and the concentration of the fragment, which from now on will be called miniVP1, were analyzed by a 12% SDS gel.

(62) For the ELISA, the 96-well plate (Costar) was covered with 25 l/well of a solution containing 300 ng of the miniVP1 fragment (generated as previously described), and incubated at 4 C. overnight. The following day, the plate is washed with water and blocked with PBS-1% BSA (w/v) for 1 hour at 37 C.

(63) At completion of the incubation of the plate, 40 L of several dilutions (serial 10-fold dilutions, starting from the undiluted up to 1:1000 dilutions) of the biological sample to be tested were added and the plate was incubated for 1 hour at 37 C. After having done 5 washes with PBS-0.1% Tween 20 (Sigma-Aldrich), by an automatic washer for ELISA microplates (ETI-System Kasher, DiaSorin), 40 L/well of a polyclonal preparation of horseradish peroxidase-conjugated goat antibodies that bind the Fc portion of human IgG (Sigma-Aldrich) were added. The plate was incubated for 45 minutes at 37 C. After 5 washes with PBS-Tween20 carried out as previously described, 40 L of substrate (1:1 solution of H.sub.2O.sub.2 and 3,3,5,5-tetramethylbenzidine, TMB substrate kit, Thermo Scientific) were added to each well, for the enzymatic reaction to occur. After about 15 minutes, the enzymatic activity was blocked by adding 40 L/well of 1N H.sub.2SO.sub.4 (Carlo Erba) and the colorimetric reaction was measured with a spectrophotometer (Model 680 Microplate Reader, Bio-Rad) at a wavelength of 450 nm.

(64) BSA antigen or another suitable antigen was introduced in each experiment as a negative control, whose O.D..sub.450 was used for detecting a possible non-specific reactivity. GRE1 monoclonal antibody was used as a positive control at a final concentration of 1 ng/A.

(65) Samples that showed a higher reactivity towards miniVP1 compared to BSA were considered positive, and thus containing JCV-GRE1-like antibodies with a neutralizing, and thus protective, activity.

(66) ELISA Assay for the In Vitro Determination of the Presence of Antibodies in Biological Samples Capable of Recognizing the Same Epitope Bound by GRE1 Monoclonal Antibody (a Variant with Competition by a Mutated VP1)

(67) In order to increase the stringency of the previous assay, another version of the experiment was set up, by adding a step of pre-incubating the sample to be tested with a VP1 mutated in the residues that are important for the binding of GRE1 to the protein.

(68) In order to obtain the VP1 protein modified in the GRE1 binding sites, and which will later act by de-selecting antibodies in the biological samples, the site-directed mutagenesis strategy was used by making some changes: since some residues to mutagenize are very close, a few residues were mutated simultaneously. First, the residues 162 and S65 were mutated by using the following primer Fw:

(69) TABLE-US-00001 (SEQIDNO:8) 5-gttttagtaagcaGCa.sup.621/AtctataGca.sup.65S/Agatac-3
and 5-tgacttactaaaacccctaagatgctcatctgg-3 (SEQ ID NO:9) as the reverse. The vector pcDNA 3.1/V5-His TOPO TA Expression Kit (Life Technologies), where the VP1 from Mad1 strain JCV had been previously cloned, was used as the template. An amplification product also containing the mutated protein was obtained by PCR. In order to remove the DNA template used for the reaction (which certainly will not contain the desired mutations), the reaction product was digested with DpnI for 4 hrs at 37 C. Thereafter, electro-competent cells were transformed with 2 L of the previously treated amplification product. Cells containing the plasmid were selected on the basis of ampicillin resistance and then checked by sequence analysis. The vector that showed the mutations inserted with the first amplification reaction was used as the template for the second amplification reaction, where the positions 127A, 130D, 131N, 133A were mutated. In order to insert these mutations,

(70) TABLE-US-00002 (SEQIDNO:10) 5-cactctaatgggcaagGa.sup.127A/Gactcat gCc.sup.130D/AGCt.sup.131N/AggtgGa.sup.133A/Gggg-3
was used as the Fw primer, and 5-cttgcccattagagtgcacattcatcaaac-3 (SEQ ID NO:11) as the reverse primer. The PCR product was digested with DpnI for 4 hrs at 37 C. Thereafter, electro-competent cells were transformed with 2 L of the previously treated amplification product. Cells containing the plasmid were selected on the basis of ampicillin resistance and then checked by sequence analysis. The VP1 that by sequence analysis exhibited all of the inserted mutations was cloned in the bacterial expression vector pET15b, in frame with 6His-tag and a thrombin cleavage site (pET-VP1mut). The mutated VP1 (VP1mut) was then purified with the same protocol as used for the purification of miniVP1.

(71) For the ELISA, the 96-well plate (Costar) was covered with 25 L/well of a solution containing 300 ng of miniVP1 (generated as previously described), and incubated at 4 C. overnight. The following day, the plate was washed with water and blocked with PBS-1% BSA (w/v) for 1 hour at 37 C. In the meanwhile, 50 g/mL of mutated VP1 was preincubated with several dilutions (serial 10-fold dilutions, starting from the undiluted up to 1:1000 dilutions) of the biological sample to be tested and incubated for 30 minutes at 37 C. At completion of the incubation of the plate, 40 L of the mix of the various serum dilutions (o other biological sample) with the mutated VP1 were added and the plate was incubated for 1 hour at 37 C. After having done 5 washes with PBS-0.1% Tween 20 (Sigma-Aldrich), by an automatic washer for ELISA microplates (ETI-System Kasher, DiaSorin), 40 L/well of a polyclonal preparation of horseradish peroxidase-conjugated goat antibodies that bind the Fc portion of human IgG (Sigma-Aldrich) were added. The plate was incubated for 45 minutes at 37 C. After 5 washes with PBS-Tween20 carried out as previously described, 40 L of substrate (1:1 solution of H.sub.2O.sub.2 and 3,3,5,5-tetramethylbenzidine, TMB substrate kit, Thermo Scientific) were added to each well, for the enzymatic reaction to occur. After about 15 minutes, the enzymatic activity was blocked by adding 40 L/well of 1N H.sub.2SO.sub.4 (Carlo Erba) and the colorimetric reaction was measured with a spectrophotometer (Model 680 Microplate Reader, Bio-Rad) at a wavelength of 450 nm.

(72) BSA antigen or another suitable antigen was introduced in each experiment as a negative control, whose O.D..sub.450 was used for detecting a possible non-specific reactivity. JCV-GRE1 monoclonal antibody was used as a positive control at a final concentration of 1 ng/L.

(73) Samples that showed a higher reactivity towards the miniVP1 peptide compared to BSA were considered positive, and thus containing JCV-GRE1-like neutralizing, and thus protective, antibodies.